Aflatoxins (AFs) are highly toxic and cancer-causing compounds, predominantly synthesized by the Aspergillus species. AFs biosynthesis is a lengthy process that requires as minimum as 30 genes grouped inside 75 kilobytes (kB) of gene clusters, which are regulated by specific transcription factors, including aflR, aflS, and some general transcription factors. This paper summarizes the status of research on characterizing structural and regulatory genes associated with AF production and their roles in aflatoxigenic fungi, particularly Aspergillus flavus and A. parasiticus, and enhances the current understanding of AFs that adversely affect humans and animals with a great emphasis on toxicity and preventive methods.

Aflatoxins (AFs) are mycotoxins, predominantly produced by Aspergillus flavus, A. parasiticus, A. nomius, and A. pseudotamarii. AFs are carcinogenic compounds causing liver cancer in humans and animals. Physical and biological factors significantly affect AF production during the pre-and post-harvest time. Several methodologies have been developed to control AF contamination, yet; they are usually expensive and unfriendly to the environment. Consequently, interest in using biocontrol agents has increased, as they are convenient, advanced, and friendly to the environment. Using non-aflatoxigenic strains of A. flavus (AF−) as biocontrol agents is the most promising method to control AFs’ contamination in cereal crops. AF− strains cannot produce AFs due to the absence of polyketide synthase genes or genetic mutation. AF− strains competitively exclude the AF+ strains in the field, giving an extra advantage to the stored grains. Several microbiological, molecular, and field-based approaches have been used to select a suitable biocontrol agent. The effectiveness of biocontrol agents in controlling AF contamination could reach up to 99.3%. Optimal inoculum rate and a perfect time of application are critical factors influencing the efficacy of biocontrol agents.

This study aimed to morphologically characterize and determine the aflatoxigenic and non-aflatoxigenic Aspergillus flavus isolates. Forty isolates of A. flavus were obtained from sweet corn kernels and soil samples collected from Kampong Raja, Rose Valley, Kea, and Klebang farms in Malaysia. They were cultured on potato dextrose agar (PDA), dichloran rose-bengal chloramphenicol (DRBC), Aspergillusflavus and Aspergillus parasiticus agar (AFPA), and coconut cream agar (CCA). Macromorphological characteristics were determined by observing the colony color and texture, while the micromorphological characteristics were determined by examining the spore color, size, structure, conidiophore structure, and vesicle shape. The production of aflatoxin was determined by direct visualization of the UV fluorescence of A. flavus colonies on CCA. Aflatoxin was qualitatively detected in 18 (45%) isolates of A. flavus using UV fluorescence screening while the remaining 22 (55%) isolates did not exhibit any aflatoxin production. The highest incidence of A. flavus (30%) and aflatoxin production (15%) was recorded in samples from Kampong Raja. On the other hand, isolates from Rose Valley (17%) and Kea (12%) were non-aflatoxigenic. Klebang recorded a 25% incidence of A. flavus in which 15% were aflatoxigenic while 10% were non-aflatoxigenic. The occurrence of aflatoxin-producing A. flavus emphasizes the need for the measure to eradicate their presence in food crops. A biological control treatment utilizing the non-aflatoxigenic strains to compete with the aflatoxigenic ones is underway. Validation of aflatoxin production through high performance liquid chromatography is also ongoing.

The pre-harvest biocontrol approach currently used includes laboratory inoculations using non-aflatoxigenic strains of Aspergillus flavus. This strategy effectively suppresses the indigenous aflatoxigenic strains and reduces aflatoxin accumulation in sweetcorn. The current in vitro study’s main objective is to determine the diametric growth rates of both Aflatoxin (AF)+ and AF− strains and improve the understanding of competitive relationships among these strains in sweetcorn (Zea mays). Sweetcorn kernels inoculated with AF+ strains only, AF− strains only, and co-inoculated with AF+ + AF− strains were investigated for aflatoxin concentrations. The diametric growth results revealed that growth rates of AF− strains at 25 and 30 °C were much greater than AF+ strains, which was in line with previous studies. The in vitro findings showed that the AKR5− and AKL34− biocontrol strains effectively inhibited the colony propagation and subsequent AFB1 contamination (up to 79%) of AF+ strains. On the other hand, the AKR1− and AKL35− were least effective in reducing AFB1 contents only by 58% and 60%, respectively. There was a significant difference (p < 0.05) in the reduction of AFB1 contents achieved by AF− strains of A. flavus. The findings of the present study indicated the reduction in AFB1 with population expressions of AF+ strains by the AF− strains and supports the notion of competitive exclusion through vigorous development and propagation of the non-aflatoxigenic fungi.

High-performance liquid chromatography (HPLC) provides a quick and efficient tool for accurately characterizing aflatoxigenic and non-aflatoxigenic isolates of Aspergillus flavus. This method also provides a quantitative analysis of AFs in Aspergillus flavus. The method’s recovery was assessed by spiking a mixture of AF at different concentrations to the testing medium. The validity of the method was confirmed using aflatoxigenic and non-aflatoxigenic strains of A. flavus. The HPLC system, coupled with a fluorescence detector and post-column photochemical reactor, showed high sensitivity in detecting spiked AFs or AFs produced by A. flavus isolates. Recovery from medium spiked with 10, 20, 60, and 80 ppb of AFs was found to be 73–86% using this approach. For AFB1 and AFB2, the limit of detection was 0.072 and 0.062 ppb, while the limit of quantification was 0.220 and 0.189 ppb, respectively. The AFB1 concentrations ranged from 0.09 to 50.68 ppb, while the AFB2 concentrations ranged between 0.33 and 9.23 ppb. The findings showed that six isolates produced more AFB1 and AFB2 than the acceptable limit of 5 ppb. The incidence of aflatoxigenic isolates of A. flavus in sweet corn and higher concentrations of AFB1 and AFB2 emphasize the need for field trials to explore their real potential for AF production in corn.